The cortical cytoskeleton supports the plasma membrane, the cell's barrier through which all small molecules pass, all signals are sensed, and all communication and attachments between cells are made. Critical to an understanding is how the cell surface is organized, especially how cells regulate attachment of the membrane to the cell cortex. This project investigates the ERM (ezrin/radixin/moesin) proteins, whose founding member, ezrin, provides a regulated linkage between the actin cytoskeleton and membrane-associated proteins in apical microvilli on polarized epithelial cells. Activated ezrin binds EBP50, a scaffolding protein with two PDZ domains that bind to many different transmembrane proteins, and a C-terminal ezrin binding domain. In addition, a cytosolic protein, EPI64 binds the first PDZ domain of EBP50. Recent work shows that EPI64 regulates the presence of microvilli on the cell surface, which is investigated in the first two aims. In the first, specific EPI64, EBP50 and ezrin mutants will be generated and their role in microvilli regulation assessed. Additional factors involved in this regulatory process will be identified and characterized. In the second, in vivo imaging of microvilli dynamics will be documented, and the role of the regulatory factors on microvilli life cycle parameters determined. In this aim, the basis for the recent finding that microvilli have subdomains will also be explored. The third aim investigates the coordination between the presence of microvilli on the cell surface and receptor-mediated endocytosis. Several studies have implicated EBP50 in endocytosis, and recent results suggest an involvement of EPI64. Analysis of endocytosis of various ligands will be determined in cells perturbed for microvillar structure. The final aim concerns the role of ezrin and moesin in the formation of the immunological synapse between a T-cell and an antigen presenting cell. Recent studies have revealed that ezrin and moesin perform distinct roles, and this aim investigates these at the molecular level. Several proteins from T cells that bind specifically to ezrin or moesin have been identified, and their role in immunological synapse formation is to be investigated. The proposed basic research has relevance to the molecular basis of disease. Many diseases can be traced to defects in plasma membrane organization and endocytic trafficking (e.g. CFTR, growth factor receptors, etc), some of which are regulated by the system under study. Moreover, ezrin is elevated in many tumors, and an elevated level of ezrin has been shown to be necessary for metastasis in some tumor cell lines.